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Expression systems for structural studies of medically important protein domains in papillomaviruses and complement

Ullmann, Christopher Graeme; (1994) Expression systems for structural studies of medically important protein domains in papillomaviruses and complement. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Prokaryotic and eukaryotic systems for the expression of recombinant proteins are extremely protein specific and these are critically reviewed in application to proteins from papillomavirus and complement. Escherichia coli and yeast a-factor expression systems were used to express the E7 protein of human papillomavirus type 16 (HPV-16) and domains in factor I of the complement system. HPV-16 E6 and E7 are transforming proteins that bind to key tumour suppressor proteins. Secondary structure predictions based on multiple sequence analysis of E6 and E7 suggested that these proteins contain zinc finger motifs of previously unknown secondary structure. E7 was expressed for structural analysis, and Fourier transform infrared (FT-IR) spectroscopy supported the predicted amount of a/? secondary structure. Secondary structure analyses of sequences and FT-IR data on peptides of another HPV-16 transforming protein, E5, showed that this protein contains primarily an a-helical secondary structure. Factor I regulates the amounts of C3b and C4b in plasma by proteolysis in the presence of cofactors. It contains five domains: a factor I module (FIM), a CD5- like domain, two low density lipoprotein receptor (LDLr) domains and a serine protease domain. As the structure and function of the FIM, LDLr1 and LDLr2 domains are unknown, these were cloned for expression studies. The yeast a-factor and the bacterial pKK233-2 systems did not yield any recombinant protein. However, the LDLr2, LDLr1/2 and FIM domains were successfully produced in fusion with the glutathione S-transferase (GST) protein using the pGEX bacterial expression system. The fusion proteins were successfully used to raise polyclonal antibodies to each domain and were also used in inhibition studies with factor I, its cofactor factor H and its substrate C3u. All three fusion proteins stimulated cleavage of C3u by factor I, possibly caused by GST. The FIM and LDLr1/2 domains have been separated from GST and purified for further work.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Expression systems for structural studies of medically important protein domains in papillomaviruses and complement
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; HPV
URI: https://discovery.ucl.ac.uk/id/eprint/10102820
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